In order to understand the molecular mechanisms underlying the regenerative ability of the urodele limb, it is important to identify regeneration-associated
proteins and to study their regulation. We have recently shown that the anti-
cytokeratin monoclonal antibody LP1K reacts strongly with newt blastemal cells, while its reactivity is restricted in normal limbs. By screening a
cDNA expression library from the newt blastema with LP1K, we have identified
cDNA clones coding for a
type II keratin (NvKII) expressed both in the mesenchyme and the specialized
wound epithelium of the blastema. While the rod domain of the
protein is highly conserved, the homology between NvKII and mammalian
type II keratins drops markedly at the N- and C-terminal regions. The expression of this
keratin was analysed by Northern blotting and RNAase protection analysis of various newt tissues, and appears to be organ specific, since it is restricted to normal and regenerating limbs and tails. In particular, we have investigated the expression of this
keratin mRNA in normal and regenerating limbs. The transcript is barely detectable in the proximal portion of the normal limb, but its level is high in the distal one. After
amputation, NvKII
mRNA is expressed both in proximal and distal blastemas, although at higher levels distally, indicating that this
keratin is regeneration associated. The NvKII transcript is detectable both in mesenchyme and in the
wound epithelium of the regenerate, while no transcript is detectable in normal epidermis. The level of NvKII
mRNA is markedly down-regulated both in normal and regenerating limbs following
intraperitoneal injection with
retinoic acid, a putative endogenous morphogen in limb regeneration.